Cognitive and negative symptoms in schizophrenia with L‐Carnosine adjuvant therapy – A randomized double‐blind placebo‐controlled study

Abstract The antioxidant L‐Carnosine is reported to improve negative and cognitive symptoms in Schizophrenia. A randomized double‐blind placebo‐controlled study was planned to study the effectiveness of adjuvant L‐Carnosine therapy in patients with Schizophrenia. 100 eligible patients with predominant negative symptoms as measured by scale for assessment of negative symptoms (SANS total score ≥ 60) and Schizophrenia diagnosis (International Classification of Disorder‐Tenth Edition, ICD‐10) were recruited. They were randomly allocated to receive a fixed dose of either 400 mg L‐Carnosine or identical placebo for 3 months and increased to 800 mg from 13th week till completion of study. Primary outcome measures assessed changes in SANS scores with L‐Carnosine at 24 weeks compared to baseline, 4 and 12 weeks. Secondary outcome measures were done to assess the improvement in cognitive symptoms (executive function, attention, and memory) at 24 weeks using subtests of NIMHANS (National Institute for Mental Health and Neurosciences) cognitive battery. Side effects were assessed using adverse events reporting form. The attention scores (p = .023) showed significant differences in patients receiving 800 mg of L‐Carnosine at the end of the study. There were no significant differences in negative symptoms in the two arms at study completion. L‐Carnosine dosing of 800 mg may be a promising agent to enhance executive functions in Schizophrenia.


| INTRODUC TI ON
L-Carnosine is a naturally occurring dipeptide (composed of β-alanine and L-histidine) which is co-localized and released with glutamate and is a widely consumed dietary supplement. β-alanine has the ability to reverse the blocking actions of GABA (Gamma-Aminobutyric acid) receptors 1 and L-histidine is involved in regulating food intake and the neurotransmitters serotonin, dopamine, and norepinephrine. 2 Cognitive deficits are predominant in patients with schizophrenia. 3 Dysregulation of N-methyl-D-aspartate receptor (NMDAR) activity and glutamate are associated with negative and cognitive symptoms in Schizophrenia and treatment with L-Carnosine is known to regulate NMDAR activity. [4][5][6] Some observational studies suggest that lower β-alanine and L-histidine may be associated with schizophrenia and that treatment with antipsychotics reversed serum histidine and alanine levels. 7 L-Carnosine is also utilized for the synthesis of histamine, which is an important neuromodulator with a role in schizophrenia and mediated by histamine receptors. 8 An early important milestone in L-Carnosine supplementation (800 mg) in autism spectrum disorder (ASD) for 8 weeks showed decreased autism severity scores compared with placebo, especially in behavior, socialization, and communication sub-scores. 9 Two randomized controlled trials evaluated L-Carnosine supplementation (2 g/day) with chronic schizophrenia on stable antipsychotic treatments and reported improvements in total scores as well as negative symptom sub-scores for schizophrenia and improvement in several cognitive domains. 10,11 Therefore, this randomized controlled trial (RCT) was conceived with the objective of studying the effectiveness of improving negative and cognitive symptoms with adjuvant L-Carnosine therapy in patients with schizophrenia.

| Study design and participants recruitment
An RCT complying with the standard protocol items recommendations for interventional trials (SPIRIT) statement was conducted. 12 This was a 24 weeks, randomized, double-blind, parallel-group, placebo-controlled study to examine the efficacy of add-on L-Carnosine in reducing negative and cognitive symptoms of schizophrenia. Patients with schizophrenia fulfilling the inclusion criteria were recruited from inpatient and outpatient departments of Schizophrenia Research Foundation, Chennai, India from June 2019 to December 2020. Written informed consent was obtained.
The study was described to each participant with a witness (usually a caregiver) present. Institutional Ethics Committee clearance was obtained before the commencement of the study. A total of 120 eligible patients were approached, out of which 100 consented to participate in the study. Patients were randomized to receive either L-Carnosine or placebo for 6 months. Study participants were assessed at baseline, 1 month, 3 months, and at the end of trial period (24 weeks from baseline). The trial was registered in the clinical trial registry in February 2019 (CTRI/2019/02/017646).

| Randomization and blinding
A total of 100 participants were randomized to receive either L-Carnosine or placebo after initial screening was completed and informed consent obtained. Allocation to treatment arm was randomly assigned in a 1:1 ratio by an independent researcher using random numbers generated. A computerized random number generator was used for the generation of randomization codes using permuted block randomization technique. The placebo tablet was not distinguishable in appearance (shape, size, color, and taste) from L-Carnosine so as to conceal treatment allocation and blinding.
Treating clinicians were also blinded to group allocation to ensure that trial treatment did not influence clinical care. All data, including biological samples, were kept in a secure location under appropriate storage conditions. The patients, rater, research assistant, laboratory assistants, and statisticians remained blinded throughout the entire trial. Table S1 depicts the time frame of enrollment, allocation, and assessments in SPIRIT format. 12

| Inclusion and exclusion criteria
The inclusion criteria were: (a) participants were required to meet

| Interventions
Patients were randomly allocated to receive either 400 mg daily of Carnosine or identical placebo for a period of 3 months. L-Carnosine dose was titrated to 800 mg in the next 3 months.
This dose was decided based on previously conducted study by Chez et al (2002). Besides, the maximum dose of L-Carnosine approved for research in India is 800 mg. Weekly telephonic interviews were conducted by research staff to ensure adherence and also to monitor for adverse events. Antipsychotic and other prescribed psychotropic medications were continued (treatment as usual) during the entire study period. Participants were instructed not to take any other vitamins and carnosine supplements, while maintaining their regular diet and physical activity during the trial.

| Clinical assessments
Sociodemographic data and relevant clinical and treatment variables were collected with the help of a semistructured proforma using medical records at baseline. Patients were assessed using scale for assessment of negative symptoms (SANS) 14

| Safety
Safety was evaluated using the adverse events reporting form 10 which includes vital signs monitoring (temperature, blood pressure, pulse rate, height, weight, and waist circumference) electrocardiogram (ECG) and laboratory tests. Adverse events and vital signs monitoring were systematically evaluated at each visit (baseline, 4, 12, and 24 weeks) using a checklist. Blood samples were obtained by venipuncture, and assessed for complete blood count, random blood glucose, renal and liver function tests at baseline, 12 and 24 weeks. ECG was also taken at baseline, 12 and 24 weeks. Table S2 depicts the time frame for assessments of parameters included in the safety monitoring schedule.

| Outcome measures
Changes in SANS and CGI scores at 24 weeks were the primary outcome measures. Secondary outcome measures included improvement in cognitive symptoms (executive function, attention, and memory) at 24 weeks assessed using subtests of NIMHANS battery.

| Statistical analysis
Demographic data were analyzed with descriptive statistics such as mean with SD for the continuous and categorical variables.
Frequencies and percentages have been reported. All analyses were based on the intention-to-treat sample and were performed using the last observation carried forward (LOCF) procedure. All continuous variables were compared across the groups using an independent t test. The dependent variables "SANS," "SAPS," and "CGI" were measured at 4-time points: baseline, 1 month, 3 months, and 6 months. Mauchly's test for the sphericity assumption was violated for "SANS" and "SAPS" scores (p < .001). To overcome the issue of sphericity, Repeated Measure anova with Greenhouse-Geisser was performed. A p < .05 was considered statistically significant.
Analysis of data was done using the Statistical Package for Social Sciences version 16. 17

| Baseline patient characteristics
Of the 100 recruited, 30 patients dropped out from the study. Of these, 11 withdrew before the first assessment and for 9 patients end line assessments could not be done due to COVID 19 lockdown restrictions. Hence, 70 patients completed the study as shown in  Table S3. Baseline SANS, SAPS, CGI scores did not differ in the two groups ( Table 1). No serious adverse events were reported in both the groups.

| Negative symptoms
The repeated measures anova was performed. Mauchly's test of sphericity indicated that the assumption of sphericity had been violated, χ 2 (2) = 71.9, p < .001, and therefore, a Greenhouse-Geisser correction was used. There is no significant difference in the SANS score between drug and placebo group (F (1.9195.9) = 0.18, p = .834) as shown in Table 1 at baseline, 4, 12, and 24 weeks. Individual domains of SANS scores did not differ in the drug and placebo group as shown in Table 2.

| Positive symptoms and CGI
Mauchly's test of sphericity indicated that the assumption of sphericity had been violated for SAPS and CGI scores and the chi-square values are χ 2 (2) = 77.6, p < .001, χ 2 (2) = 37.1, p < .001, respectively, and therefore, a Greenhouse-Geisser correction was used. Both SAPS and CGI scores did not differ in the two groups ( Table 1).

| Cognitive symptoms
The L-Carnosine treated group showed a significantly greater improvement only in trail making tests B when compared to placebo group (p = .023) as shown in Table 3 and Figure 2. There are no TA B L E 1 Repeated measure analysis for comparison of the two treatment effects on SANS, SAPS, CGI scores.

| Adverse events
During the entire study, 22 adverse effects were recorded. The frequency of side effects did not differ significantly between two groups as shown in Table 4. No major adverse event was reported and they did not account for the dropouts.

| DISCUSS ION
L-Carnosine is widely used as an antioxidant and neutraceutical agent. Studies in L-carnosine had used variable dosing of 200 mg-2 g across schizophrenia and autism spectrum disorder.
A recent study from Tehran proposed that this drug improves negative symptoms and cognition and highlighted that a 2 g dose of L-Carnosine is necessary to exert any glutamatergic or NMDA modulatory effects in schizophrenia. 10,11 On the contrary, our 24-weeks study found no change from baseline on the negative symptoms domain scores. This contrasting finding could be due to the dosing patterns (800 mg vs. 2 g), duration of study (24 vs. 8 weeks) and illness-related variables such as chronicity, long duration of illness, and treatment resistance seen in our study population. Indian research on the optimal dose of L-Carnosine is lacking.
In the Indian setting, L-Carnosine was commercially available as 200 mg and for study purpose the drug manufacturer had regulatory agency approval for 400 mg tablets (drug and placebo). This was the first Indian study to examine the efficacy and safety of L-Carnosine so the study drug dosing was fixed at 400 mg for 12 weeks and increased to 800 mg till the completion of the study.  worthy. Therefore, a well-designed study using optimal dosing of 1.5-2 g for longer duration can eliminate the placebo response ideally.

TA B L E 3
Comparison of cognitive domains between the two groups using t tests.
F I G U R E 2 TRAIL B mean plot between drug and the placebo group.
In summary, controlling for combined effects of confounding variables such as age and gender, matching illness severity (SANS score > 60) in the two groups, concurrent antipsychotic medications, chronic nature of the illness, and placebo response were insufficient to produce the desired results.
A larger multicenter longitudinal study using higher doses of Lcarnosine 2 g or more should be planned to explore the desired efficacy of the drug in improving all domains of cognition and negative symptoms.
This current study and all previous studies using L-Carnosine in schizophrenia did not report any major adverse events.

| LIMITATI ON S AND FUTURE DIREC TION
This was a double-blind, placebo-controlled, prospective study with adequate sample size. However, the low dose of 400 or 800 mg has been the major limitation. A multisite study using a differential dosing of L-Carnosine (1.5-2 g), longer follow up, utility of newer scales such as BNSS (Brief Negative Symptoms Scale) or CAINS (Clinical Assessment Interview for Negative Symptoms) and Anticholinergic burden assessment may throw more conclusions on the efficacy of the drug. Additionally, prospectively measuring serum concentrations of alanine and glutamate in an L-Carnosine study can also establish these as putative inflammatory biomarkers in schizophrenia.

| CON CLUS ION
L-Carnosine add-on therapy (800 mg) in schizophrenia has shown improvement in executive function although negative symptoms did not improve.

ACK N OWLED G M ENTS
All patients and their families who consented to participate in the study.

FU N D I N G I N FO R M ATI O N
This study was supported by the Shield Health Care.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data cannot be shared publicly because of privacy/ethical restrictions.

CO N FLI C T O F I NTER E S T S TATEM ENT
There are no conflicts of interest in relation to the subject of this study. Foot and joint pain 0 (0) 0 (0)